136 resultados para Complemented Subgroups
Resumo:
Although cerebral palsy (CP) is the most common cause of motor deficiency in young children, it occurs in only 2 to 3 per 1000 live births. In order to monitor prevalence rates, especially within subgroups (birthweight, clinical type), it is necessary to study large populations. A network of CP surveys and registers was formed in 14 centres in eight countries across Europe. Differences in prevalence rates of CP in the centres prior to any work on harmonization of data are reported. The subsequent process to standardize the definition of CP, inclusion/exclusion criteria, classification, and description of children with CP is outlined. The consensus that was reached on these issues will make it possible to monitor trends in CP rate, to provide a framework for collaborative research, and a basis for services planning among European countries.
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Investigations of the factor structure of the Alcohol Use Disorders Identification Test (AUDIT) have produced conflicting results. The current study assessed the factor structure of the AUDIT for a group of Mentally Disordered Offenders (MDOs) and examined the pattern of scoring in specific subgroups. The sample comprised 2005 MDOs who completed a battery of tests including the AUDIT. Confirmatory factor analyses revealed that a two-factor solution – alcohol consumption and alcohol-related consequences – provided the best data fit for AUDIT scores. A three-factor solution provided an equally good fit, but the second and third factors were highly correlated and a measure of parsimony also favoured the two-factor solution. This study provides useful information on the factor structure of the AUDIT amongst a large MDO population, while also highlighting the difficulties associated with the presence of people with mental health problems in the criminal justice system.
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The first report of time-resolved resonance Raman (TR(3)) scattering in a supercritical fluid is presented. TR(3) spectra of the lowest triplet excited state (T-1) of anthracene in supercritical (SC) CO2 have been obtained over the pressure range 90-500 bar. These data have been complemented by conventional flash photolysis measurements of the excited state lifetime, transient absorbance difference, and fluorescence spectra over a similar pressure range. The spectroscopic data show systematic changes with increasing pressure; the Delta A spectra of the TI state recorded at two different temperatures display a red shift with increasing fluid pressure, which is in agreement with earlier work carried out over a smaller range of pressures. Similar shifts in the fluorescence are also observed. The vibrational frequencies of the T-1 state of anthracene are found to be relatively insensitive to applied pressure; indeed, the transient bands are readily identified by comparison with resonance Raman (RR) spectra of the T-1 state in cyclohexane solution. Small but well-defined shifts to lower cm(-1) with increasing pressure are observed in some of the vibrational bands of SC COE. The most marked change in the excited state Raman spectra is that the intensity of the T-1 anthracene features, relative to those of CO2, increases with applied pressure. The information which each of the above spectroscopic methods gives on the question of how pressure changes affect the structure and local environment of the excited state probe molecule in the SCF is discussed. Possible explanations for the observed increase in RR band intensities in terms of increased resonance Raman enhancement arising from the spectral shifts and/or the increased solubility of anthracene in CO2 with increasing pressure are also considered.
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This edition of Milton’s Epistolarum Familiarium Liber Unus and of his Uncollected Letters, will appear as 672 pp. of The Complete Works of John Milton Volume XI, eds. Gordon Campbell and Edward Jones (Oxford University Press, forthcoming 2016). A diplomatic Latin text and a new facing English translation are complemented by a detailed Introduction and commentary that situate Milton’s Latin letters in relation to the classical, pedagogical and essentially humanist contexts at the heart of their composition. Now the art of epistolography advocated and exemplified by Cicero and Quintilian and embraced by Renaissance pedagogical manuals is read through a humanist filter whereby, via the precedent (and very title) of Epistolae Familiares, the Miltonic Liber is shown to engage with a neo-Latin re-invention of the classical epistola that had come to birth in quattrocento Italy in the letters of Petrarch and his contemporaries. At the same time the Epistolae are seen as offering fresh insight into Milton’s views on education, philology, his relations with Italian literati, his blindness, the poetic dimension of his Latin prose, and especially his verbal ingenuity as the ‘words’ of Latin ‘Letters’ become a self-conscious showcasing of etymological punning on the ‘letters’ of Latin ‘words’. The edition also announces several new discoveries, most notably its uncovering and collation of a manuscript of Henry Oldenburg’s transcription (in his Liber Epistolaris held in Royal Society, London) of Milton’s Ep. Fam. 25 (to Richard Jones). Oldenburg’s transcription (from the original sent to his pupil Jones) is an important find, given the loss of all but two of the manuscripts of Milton’s original Latin letters included in the 1674 volume. The edition also presents new evidence in regard to Milton’s relationships with the Italian philologist Benedetto Buonmattei, the Greek humanist Leonard Philaras, the radical pastor Jean Labadie (and the French church of London), and the elusive Peter Heimbach.
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Introduction: Basal-like breast cancers (BL-BCa) have the worst prognosis of all subgroups of this disease. Hyaluronan (HA) and the HA receptor CD44 have a long-standing association with cell invasion and metastasis of breast cancer. The purpose of this study was to establish the relation of CD44 to BL-BCa and to characterize how HA/CD44 signaling promotes a protease-dependent invasion of breast cancer (BrCa) cells.
Methods: CD44 expression was determined with immunohistochemistry (IHC) analysis of a breast cancer tissue microarray (TMA). In vitro experiments were performed on a panel of invasive BL-BCa cell lines, by using quantitative polymerase chain reaction (PCR), immunoblotting, protease activity assays, and invasion assays to characterize the basis of HA-induced, CD44-mediated invasion.
Results: Expression of the hyaluronan (HA) receptor CD44 associated with the basal-like subgroup in a cohort of 141 breast tumor specimens (P = 0.018). Highly invasive cells of the representative BL-BCa cell line, MDA-MB-231 (MDA-MB-231Hi) exhibited increased invasion through a basement membrane matrix (Matrigel) and collagen. In further experiments, HA-induced promotion of CD44 signaling potentiated expression of urokinase plasminogen activator (uPA) and its receptor uPAR, and underpinned an increased cell-associated activity of this serine protease in MDA-MB-231Hi and a further BL-BCa cell line, Hs578T cells. Knockdown of CD44 attenuated both basal and HA-stimulated uPA and uPAR gene expression and uPA activity. Inhibition of uPA activity by using (a) a gene-targeted RNAi or (b) a small-molecule inhibitor of uPA attenuated HA-induced invasion of MDA-MB-231Hi cells through Matrigel. HA/CD44 signaling also was shown to increase invasion of MDA-MB-231 cells through collagen and to potentiate the collagen-degrading activity of MDA-MB-231Hi cells. CD44 signaling was subsequently shown to upregulate expression of two potent collagen-degrading enzymes, the cysteine protease cathepsin K and the matrix metalloprotease MT1-MMP. RNAi- or shRNA-mediated depletion of CD44 in MDA-MB-231Hi cells decreased basal and HA-induced cathepsin K and MT1-MMP expression, reduced the collagen-degrading activity of the cell, and attenuated cell invasion through collagen. Pharmacologic inhibition of cathepsin K or RNAi-mediated depletion of MT1-MMP also attenuated MDA-MB-231Hi cell invasion through collagen.
Conclusion: HA-induced CD44 signaling increases a diverse spectrum of protease activity to facilitate the invasion associated with BL-BCa cells, providing new insights into the molecular basis of CD44-promoted invasion.
Resumo:
Evidence is accumulating that vitamin D may be protective against carcinogenesis, although exceptions have been observed for some digestive tract neoplasms. The aim of the present study was to explore the association between dietary vitamin D and related nutrients and the risk of oesophageal adenocarcinoma and its precursor conditions, Barrett's oesophagus and reflux oesophagitis. In an all-Ireland case-control study conducted between March 2002 and July 2005, 218 oesophageal adenocarcinoma patients, 212 Barrett's oesophagus patients, 208 reflux oesophagitis patients and 252 population-based controls completed a 101-item FFQ, and provided lifestyle and demographic information. Multiple logistic regression analysis was applied to examine the association between dietary intake and disease risk. Oesophageal adenocarcinoma risk was significantly greater for individuals with the highest compared with the lowest tertile of vitamin D intake (OR 1·99, 95 % CI 1·03, 3·86; P for trend = 0·02). The direct association could not be attributed to a particular vitamin D food source. Vitamin D intake was unrelated to Barrett's oesophagus and reflux oesophagitis risk. No significant associations were observed for Ca or dairy intake and oesophageal adenocarcinoma, Barrett's oesophagus or reflux oesophagitis development. High vitamin D intake may increase oesophageal adenocarcinoma risk but is not related to reflux oesophagitis and Barrett's oesophagus. Ca and dairy product intake did not influence the development of these oesophageal lesions. These findings suggest that there may be population subgroups at an increased risk of oesophageal adenocarcinoma if advice to improve vitamin D intake from foods is implemented. Limited work has been conducted in this area, and further research is required.
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WbaP is a membrane enzyme that initiates O antigen synthesis in Salmonella enterica by catalysing the transfer of galactose 1-phosphate (Gal-1-P) onto undecaprenyl phosphate (Und-P). WbaP possesses at least three predicted structural domains: an N-terminal region containing four transmembrane helices, a large central periplasmic loop, and a C-terminal domain containing the last transmembrane helix and a large cytoplasmic tail. In this work, we investigated the contribution of each region to WbaP function by constructing a series of mutant WbaP proteins and using them to complement O antigen synthesis in DeltawbaP mutants of S. enterica serovars Typhi and Typhimurium. Truncated forms of WbaP lacking the periplasmic loop exhibited altered chain-length distributions in O antigen polymerization, suggesting that this central domain is involved in modulating the chain-length distribution of the O polysaccharide. The N-terminal and periplasmic domains were dispensable for complementation of O antigen synthesis in vivo, suggesting that the C-terminal domain carries the sugar-phosphate transferase activity. However, despite the fact that they complemented the synthesis of O antigen in the DeltawbaP mutant in vivo, membrane extracts containing WbaP derivatives without the N-terminal domain failed to transfer radioactive Gal from UDP-Gal into a lipid-rich fraction. These results suggest that the N-terminal region of WbaP, which contains four transmembrane domains, is essential for the insertion or stability of the protein in the bacterial membrane. We propose that the domain structure of WbaP enables this protein not only to function in the transfer of Gal-1-P to Und-P but also to establish critical interactions with additional proteins required for the correct assembly of O antigen in S. enterica.
Resumo:
Burkholderia cenocepacia is a gram-negative, non-spore-forming bacillus and a member of the Burkholderia cepacia complex. B. cenocepacia can survive intracellularly in phagocytic cells and can produce at least one superoxide dismutase (SOD). The inability of O2- to cross the cytoplasmic membrane, coupled with the periplasmic location of Cu,ZnSODs, suggests that periplasmic SODs protect bacteria from superoxide that has an exogenous origin (for example, when cells are faced with reactive oxygen intermediates generated by host cells in response to infection). In this study, we identified the sodC gene encoding a Cu,ZnSOD in B. cenocepacia and demonstrated that a sodC null mutant was not sensitive to a H2O2, 3-morpholinosydnonimine, or paraquat challenge but was killed by exogenous superoxide generated by the xanthine/xanthine oxidase method. The sodC mutant also exhibited a growth defect in liquid medium compared to the parental strain, which could be complemented in trans. The mutant was killed more rapidly than the parental strain was killed in murine macrophage-like cell line RAW 264.7, but killing was eliminated when macrophages were treated with an NADPH oxidase inhibitor. We also confirmed that SodC is periplasmic and identified the metal cofactor. B. cenocepacia SodC was resistant to inhibition by H2O2 and was unusually resistant to KCN for a Cu,ZnSOD. Together, these observations establish that B. cenocepacia produces a periplasmic Cu,ZnSOD that protects this bacterium from exogenously generated O2- and contributes to intracellular survival of this bacterium in macrophages.
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Translocation of lipid-linked oligosaccharide (LLO) intermediates across membranes is an essential but poorly understood process in eukaryotic and bacterial glycosylation pathways. Membrane proteins defined as translocases or flippases are implicated to mediate the translocation reaction. The membrane protein Wzx has been proposed to mediate the translocation across the plasma membrane of lipopolysaccharide (LPS) O antigen subunits, which are assembled on an undecaprenyl pyrophosphate lipid carrier. Similarly, PglK (formerly WlaB) is a Campylobacter jejuni-encoded ABC-type transporter proposed to mediate the translocation of the undecaprenylpyrophosphate-linked heptasaccharide intermediate involved in the recently identified bacterial N-linked protein glycosylation pathway. A combination of genetic and carbohydrate structural analyses defined and characterized flippase activities in the C. jejuni N-linked protein glycosylation and the Escherichia coli LPS O antigen biosynthesis. PglK displayed relaxed substrate specificity with respect to the oligosaccharide structure of the LLO intermediate and complemented a wzx deficiency in E. coli O-antigen biosynthesis. Our experiments provide strong genetic evidence that LLO translocation across membranes can be catalyzed by two distinct proteins that do not share any sequence similarity.
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Background: Bacteria employ complex transcriptional networks involving multiple genes in response to stress, which is not limited to gene and protein networks but now includes small RNAs (sRNAs). These regulatory RNA molecules are increasingly shown to be able to initiate regulatory cascades and modulate the expression of multiple genes that are involved in or required for survival under environmental challenge. Despite mounting evidence for the importance of sRNAs in stress response, their role upon antibiotic exposure remains unknown. In this study, we sought to determine firstly, whether differential expression of sRNAs occurs upon antibiotic exposure and secondly, whether these sRNAs could be attributed to microbial tolerance to antibiotics.
Results: A small scale sRNA cloning strategy of Salmonella enterica serovar Typhimurium SL1344 challenged with half the minimal inhibitory concentration of tigecycline identified four sRNAs (sYJ5, sYJ20, sYJ75 and sYJ118) which were reproducibly upregulated in the presence of either tigecycline or tetracycline. The coding sequences of the four sRNAs were found to be conserved across a number of species. Genome analysis found that sYJ5 and sYJ118 mapped between the 16S and 23S rRNA encoding genes. sYJ20 (also known as SroA) is encoded upstream of the tbpAyabKyabJ operon and is classed as a riboswitch, whilst its role in antibiotic stress-response appears independent of its riboswitch function. sYJ75 is encoded between genes that are involved in enterobactin transport and metabolism. Additionally we find that the genetic deletion of sYJ20 rendered a reduced viability phenotype in the presence of tigecycline, which was recovered when complemented. The upregulation of some of these sRNAs were also observed when S. Typhimurium was challenged by ampicillin (sYJ5, 75 and 118); or when Klebsiella pneumoniae was challenged by tigecycline (sYJ20 and 118).
Conclusions: Small RNAs are overexpressed as a result of antibiotic exposure in S. Typhimurium where the same molecules are upregulated in a related species or after exposure to different antibiotics. sYJ20, a riboswitch, appears to possess a trans-regulatory sRNA role in antibiotic tolerance. These findings imply that the sRNA mediated response is a component of the bacterial response to antibiotic challenge.
Resumo:
Polymyxin B-sensitive mutants in Burkholderia vietnamiensis (Burkholderia cepacia genomovar V) were generated with a mini-Tn5 encoding tetracycline resistance. One of the transposon mutants had an insertion in the norM gene encoding a multi-drug efflux protein. Expression of B. vietnamiensis norM in an Escherichia coli acrAB deletion mutant complemented its norfloxacin hypersensitivity, indicating that the protein functions in drug efflux. However, no effect on antibiotic sensitivity other than sensitivity to polymyxin B was observed in the B. vietnamiensis norM mutant. We demonstrate that increased polymyxin sensitivity in B. vietnamiensis was associated with the presence of tetracycline in the growth medium, a phenotype that was partially suppressed by expression of the norM gene.
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We investigated the involvement of Tol proteins in the surface expression of lipopolysaccharide (LPS). tolQ, -R, -A and -B mutants of Escherichia coli K-12, which do not form a complete LPS-containing O antigen, were transformed with the O7+ cosmid pJHCV32. The tolA and tolQ mutants showed reduced O7 LPS expression compared with the respective isogenic parent strains. No changes in O7 LPS expression were found in the other tol mutants. The O7-deficient phenotype in the tolQ and tolA mutants was complemented with a plasmid encoding the tolQRA operon, but not with a similar plasmid containing a frameshift mutation inactivating tolA. Therefore, the reduction in O7 LPS was attributed to the lack of a functional tolA gene, caused either by a direct mutation of this gene or by a polar effect on tolA gene expression exerted by the tolQ mutation. Reduced surface expression of O7 LPS was not caused by changes in lipid A-core structure or downregulation of the O7 LPS promoter. However, an abnormal accumulation of radiolabelled mannose was detected in the plasma membrane. As mannose is a sugar unique to the O7 subunit, this result suggested the presence of accumulated O7 LPS biosynthesis intermediates. Attempts to construct a tolA mutant in the E. coli O7 wild-type strain VW187 were unsuccessful, suggesting that this mutation is lethal. In contrast, a polar tolQ mutation affecting tolA expression in VW187 caused slow growth rate and serum sensitivity in addition to reduced O7 LPS production. VW187 tolQ cells showed an elongated morphology and became permeable to the membrane-impermeable dye propidium iodide. All these phenotypes were corrected upon complementation with cloned tol genes but were not restored by complementation with the tolQRA operon containing the frameshift mutation in tolA. Our results demonstrate that the TolA protein plays a critical role in the surface expression of O antigen subunits by an as yet uncharacterized involvement in the processing of O antigen.
Resumo:
The O-repeating unit of the Escherichia coli O7-specific lipopolysaccharide is made of galactose, mannose, rhamnose, 4-acetamido-4,6-dideoxyglucose, and N-acetyglucosamine. We have recently characterized the genes involved in the biosynthesis of the sugar precursor GDP-mannose occurring in the E. coli O7:K1 strain VW187 (C. L. Marolda and M. A. Valvano, J. Bacteriol. 175:148-158, 1993). In the present study, we identified and sequenced the rfbBDAC genes encoding the enzymes for the biosynthesis of another precursor, dTDP-rhamnose. These genes are localized on the upstream end of the rfbEcO7 region, and they are strongly conserved compared with similar genes found in various enteric and nonenteric bacteria. Upstream of rfbB we identified a DNA segment containing the rfb promoter and a highly conserved untranslated leader sequence also present in the promoter regions of other surface polysaccharide gene clusters. Also, we have determined that rfbB and rfbA have homologs, rffG (o355) and rffH (o292), respectively, located on the rff cluster, which is involved in the synthesis of enterobacterial common antigen. We provide biochemical evidence that rffG and rffH encode dTDP-glucose dehydratase and glucose-1-phosphate thymidylyltransferase activities, respectively, and we also show that rffG complemented the rfbB defect in the O7+ cosmid pJHCV32. We also demonstrate that rffG is distinct from rffE and map the rffE gene to the second gene of the rff cluster.
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We report that rfe mutants of wild-type strains of Escherichia coli O7, O18, O75, and O111 did not express O-specific polysaccharide unless the rfe mutation was complemented by a cloned rfe gene supplied in a plasmid. The O polysaccharides in these strains are known to have N-acetylglucosamine (GlcNAc) in their O repeats. In addition, in vitro transferase assays with bacterial membranes from either the O7 wild-type strain or its isogenic rfe mutant showed that GlcNAc is the first carbohydrate added onto the lipid acceptor in the assembly of the O7 repeat and that this function is inhibited by tunicamycin. Our results indicate that the rfe gene product is a general requirement for the synthesis of O polysaccharides containing GlcNAc.
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We recently reported a novel genetic locus located in the sbcB-his region of the chromosomal map of Escherichia coli K-12 which directs the expression of group 6-positive phenotype in Shigella flexneri lipopolysaccharide, presumably due to the transfer of O-acetyl groups onto rhamnose residues of the S. flexneri O-specific polysaccharide (Z. Yao, H. Liu, and M. A. Valvano, J. Bacteriol. 174:7500-7508, 1992). In this study, we identified the genetic region encoding group 6 specificity as part of the rfb gene cluster of E. coli K-12 strain W3110 and established the DNA sequence of most of this cluster. The rfbBDACX block of genes, located in the upstream region of the rfb cluster, was found to be strongly conserved in comparison with the corresponding region in Shigella dysenteriae type 1 and Salmonella enterica. Six other genes, four of which were shown to be essential for the expression of group 6 reactivity in S. flexneri serotypes Y and 4a, were identified downstream of rfbX. One of the remaining two genes showed similarities with rfc (O-antigen polymerase) of S. enterica serovar typhimurium, whereas the other, located in the downstream end of the cluster next to gnd (gluconate-6-phosphate dehydrogenase), had an IS5 insertion. Recently, it has been reported that the IS5 insertion mutation (rfb-50) can be complemented, resulting in the formation of O16-specific polysaccharide by E. coli K-12 (D. Liu and P. R. Reeves, Microbiology 140:49-57, 1994). We present immunochemical evidence suggesting that S. flexneri rfb genes also complement the rfb-50 mutation; in the presence of rfb genes of E. coli K-12, S. flexneri isolates express O16-specific polysaccharide which is also acetylated in its rhamnose residues, thereby eliciting group 6 specificity.